Protein Folding and Disease: The Path from Bench to Bedside

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Vicki Glaser

Cell biologists are exploring and exploiting protein-folding pathways as a source of drug targets for diseases involving disruption of protein homeostatis, or proteostasis.

Such diseases affect proteome expression, protein function, and proteome maintenance. The “American Society for Cell Biology” (ASCB) annual meeting featured presentations that illustrated how managing the protein fold can modify local proteostasis activity to intervene in protein folding, degradation, and function across a range of human diseases including cancer, cystic fibrosis, and neurodegenerative diseases.

One researcher who spoke at the meeting was Jeffery Kelly, Lita Annenberg Hazen Professor of Chemistry and chairman, department of molecular and experimental medicine at the Scripps Institute. Dr. Kelly’s lab studies proteostasis from the perspective of the competition between protein folding, misfolding, and aggregation, and the link between diseases and protein misfolding coupled to degradation or aggregation.

His team then designs small molecules to enhance biological protein maintenance mediated by the proteostatis network, for example, by enhancing protein folding and trafficking or promoting clearance of misfolding- or aggregation-prone proteins. A complementary chemical approach creates a small molecule that binds to the native state of a specific misfolding-prone protein to prevent misfolding and aggregation. The latter strategy has led to the discovery of a first-in-class small molecule drug to treat gain-of-toxic-function transthyretin amyloid diseases associated with neuro- and cardio-degeneration.

Specifically, Dr. Kelly described the development of the drug tafamidis (Vyndaqel, FoldRx/Pfizer), which recently received approval by the EMA and is under review by the U.S. FDA. This drug, developed using structure-based design principles, binds to a site at the dimer-dimer interface of the native tetrameric structure of the protein transthyretin.

In the gain-of-toxic-function transthyretin amyloid diseases, aberrant dissociation of the tetramer allows the misfolded monomers to aggregate outside the cell. In this autosomal dominant disease, the mutant and wild-type (wt) subunits produced by heterozygotes are able to form heterotetramers that can evade the cellular quality control system and are secreted from the cell. However, these tetramers are less stable than their wt-wt counterparts and more readily dissociate once secreted, facilitating toxic amyloid formation.

Dr. Kelly’s group targeted the weaker of the dimer-dimer interfaces and crafted a small molecule that binds with high affinity and specificity and prevents the tetramer from dissociating outside the cell, thereby inhibiting protein aggregation and amyloid formation.

Amyloid pathology plays a key role in several degenerative diseases, including peripheral nervous system disorders and cardiomyopathies. “Tafamidis is the only approved drug targeting the underlying cause of an amyloid disease,” reports Dr. Kelly. The potential for these types of drugs “is huge,” he adds. “Protein misfolding or aggregation is a component of diseases that “span virtually every therapeutic area, from eye, to kidney, metabolic, heart, and neurodegenerative diseases.”

Maintaining Proteostasis

Alfred Goldberg, Ph.D., professor of cell biology at Harvard Medical School, traced the path by which bortezomib (Velcade, Millennium: The Takeda Oncology Company) became an approved drug for the treatment of multiple myeloma.

Bortezomib selectively inhibits the active site of the 26S proteasome, a proteolytic complex that functions as a sort of “trash collector” inside cells and is entrusted with the job of degrading proteins marked for destruction through the addition of ubiquitin chains. The 26S proteasome utilizes ATP to unfold proteins, sending them off for subsequent digestion into small peptides.

Different types of errors in this proteasome maintenance pathway can cause disease, including failure to recognize misfolded proteins that may then aggregate or be dysfunctional, or excessive proteolytic activity that results in the destruction of proteins necessary for normal cell function.

The initial work that led to the discovery of bortezomib was focused on identifying a research tool that could be used to explore and understand protease inhibition—a proteasome inhibitor capable of blocking proteolysis.

This led to the discovery of other activities for these inhibitors, including anti-inflammatory and antineoplastic effects related to activation of the NFκB pathway. The primary target for this drug discovery program early on was the excessive protein breakdown associated with muscle atrophy and cachexia.

NFκB is a critical transcription factor in inflammation and also plays a key role in cell growth in myeloma cells. The ability of bortezomib to intervene in these pathways and also to inhibit the selective degradation of misfolded proteins—and specifically, abnormal immunoglobulins—resulted in rapid progression of the compound through clinical development and approval for marketing in the U.S. after Phase II trials.

Targeting the Underlying Defects

The take-home message Fredrick van Goor, Ph.D., scientist at Vertex Pharmaceuticals, delivered was “to target the underlying defect” of a disease and not its symptoms.

In the case of VX-770, which has completed Phase III trials and is under FDA review, and VX-809, which is in late-stage clinical studies, this involves targeting the multiple possible mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene that can result in different types of defects underlying CF pathology.

“We developed two different types of drugs tailored to the different types of defects, protein quantity and function: protein correctors targeting quantity, and potentiators targeting function,” both with the therapeutic goal of increasing chloride ion-channel gating activity, said Dr. van Goor.

Although the first description of the loss of chloride transport defect underlying CF was reported in 1983, and the identification of the CFTR gene in 1989, the road to the discovery and development of therapeutic agents targeting the underlying defects in CF was long and challenging.

Protein misfolding is only one aspect of what can go wrong in CF, and the pathologic underpinnings of the disease and the factors that determine its severity are more complex than a single mutation or even a single mechanism.

Early on, the researchers recognized the need to develop a combination therapy composed of a CFTR corrector that would allow for more full-length, correctly folded CFTR to reach the cell surface, and a CFTR potentiator that would enhance CFTR activity once the chloride ion channel was activated.

Approximately 1,800 mutations have been associated with CF, according to Dr. van Goor. By grouping them into three categories—gating defects affecting the opening and closing of the chloride channel, chloride transport defects in which some functional CFTR makes its way to the cell surface enabling residual protein function, and protein trafficking defects that are mainly caused by deletion mutations and prevent functional CFTR from getting to the cell surface—the researchers were able to develop treatments aimed at overcoming each defect.